AUTOIMMUNE AND INFLAMMATORY RESPONSES AFFECTING THE RETINA Most of these studies emphasize mouse models of autoimmune uveitis developed in our laboratory, (i) experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal Ag IRBP, and (ii) spontaneous uveitis, which develops in R161H mice that express a transgenic T cell receptor (TCR) specific for the IRBP epitope encoded by residues 161-180, on the B10.RIII background. To better study basic mechanisms, we have backcrossed numerous gene-manipulated strains onto the B10.RIII/R161H background. (1) Recent findings in R161H mice indicated that gut commensal flora is necessary for development of spontaneous uveitis. Interestingly, while depletion of gut flora by antibiotic treatment starting before birth inhibited disease development in the spontaneous model, it had no effect on the (immunization induced) EAU model. However, a short-term antibiotic treatment did afford measurable protection in the EAU model. Further experiments indicated that antibiotic treatment depletes intraepithelial lymphocytes (IEL), which are known to be microbiota-dependent and may which we hypothesize to have regulatory function in uveitis. We are currently working on demonstrating disease-relevant regulatory function of IELs and connecting specific microbiota to their maintenance in the gut. (Salvador et al., in preparation). (2) The role of various cytokines in pathogenesis and regulation of uveitis is being studied using both the classical and the spontaneous uveitis models, with emphasis on the balance between Th1 and Th17 responses, and their control by T-regulatory (Treg) cells. Although previous studies indicated a critical role for the Th17 lineage cytokines in pathogenesis of immunization-induced EAU, crossing R161H mice to IFN-g deficient or IL-17 deficient mice unexpectedly revealed a major role for IFN- in the spontaneous disease. Using mice deficient in both IL-17 and IFN- demonstrated that disease is still induced, but is now mediated by distinct effector cytokine profile. The results implicated GM-CSF as a critical pathogenic cytokine, which may drive disease in the absence of both IFN- and IL-17 (S Bing et al, manuscript in preparation). On the other hand, IL-22 appears to have a protective role, which may at least in part be due to be due to a neuroprotective effect of IL-22. Since neurons do not seem to express a receptor for IL-22, this is likely an indirect effect that requires other retinal cells (Mattapallil et al, manuscript submitted). Although IL-17 is a proinflammatory cytokine in EAU, it also appears to act as a negative regulator of the Th17 differentiation pathway. Data indicate that, by binding to the IL-17R on activated T cells, IL17 elicits a regulatory pathway that inhibits production of other Th17-related cytokines, including, importantly, GM-CSF. We speculate that the loss of this regulatory pathway in uveitis patients treated with anti-IL-17 neutralizing antibody (Secukinumab) may underlie the unexpectedly disappointing effects of this therapy (WP Chong et al., in preparation). In collaboration with Dr. Benjamin Sredni of Bar Ilan University, Israel, we showed that the immunomodulatory organotellurium compound, AS-101, ameliorates experimental autoimmune uveitis by regulating Th1 and Th17 responses and inducing Treg cells. (S Bing et al, manuscript in preparation). Finally, in collaboration with the group of Dr. Charles Egwuagu (NEI, LI, project # EY000350) it was found that IL-12p35 is an effective immunoregulatory agent. Treatment with this engineered cytokine induced IL-10 and IL-35-expressing regulatory B cells and T cells, inhibited the expansion and trafficking of pathogenic Th17 and Th1 cells, and ameliorated EAU (Dambuza et al, Nature Communications) and EAE (J Choi et al., Frontiers in Immunology). (3) Vitamin (VitA) derivatives are necessary for functional activation of immune cells (published literature). We previously demonstrated the importance of Vitamin A (VitA) and its metabolite, retinoic acid, in ocular immune privilege. Using mice made VitA deficient (VAD), we found that T cell effector function that was acquired before onset of VAD is maintained in the VAD host. These findings may have clinical implications in geographical regions where dietary VitA is limiting. (Horai, Zhou et al, in preparation). (4) In collaboration with Drs. Daniela Verthelyi and her group at FDA/CBER we are studying ocular effects of neonatal infection with Zika virus or rVSVG-ZEBOV-GP (vesicular stomatitis pseudovirus expressing Ebola virus glycoprotein). The data suggest that ocular immune privilege plays a role in promoting persistence of infectious agents within the eye and brain, where they are protected from elimination by systemic immune mechanisms. These studies have the potential to help elucidate the mechanisms underlying the damage that these viral agents cause to the eyes and aid in the development of effective therapeutics (Manangeeswaran M et al., Emerging Microbes & Infections 2018 and manuscript submitted for publication). EFFECTS OF INNATE IMMUNE RESPONSES ON AUTOIMMUNITY: Cellular and molecular elements of the innate immune system can affect immunopathogenic processes directly as well as indirectly, by affecting adaptive immunity. (1) The transcription factor STAT3 has been defined as necessary for the induction of IL-17 production by adaptive and innate cells. Our recent studies indicate that some types of innate lymphocytes, including NKT cells, innate-like CD8 cells and CD4/CD8 double-negative T cells, are able to produce IL-17 via a pathway that does not require STAT3. In vivo experiments suggest that this pathway may have a function in protecting from infections at the ocular surface. (St. Leger, Hansen et al, JEM, Feb 2018). (2) Innate immunity receptors have a major role in controlling susceptibility to autoimmune uveitis. A collaborative study with Dr. Holly Rosenzweig lab at OHSU, Portland, revealed that, unexpectedly, NOD2, usually thought of as an inflammation-promoting receptor, limits autoimmunity to the neuroretina. This appears to occur through a T cell intrinsic mechanism, by downrgulating IL-17 production from T cells, but not through conventional APC priming. This study uncovers a hitherto unrecognized role for Nod2 as an inherent genetic modifier of T cell function in uveitis.. (Manuscript submitted). (3) A collaborative study with Dr. Feng Lin at the Cleveland Clinic, Cleveland, Ohio, revealed that the Complement component C4 regulates T cell responses in a cell-intrinsic fashion (Lin et al., Frontiers in Immunology, 2017). MUCOSAL IMMUNE RESPONSES AT THE OCULAR SURFACE AND OCULAR SURFACE MICROBIOME (1) Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but presence of ocular surface microbiome was contentious. Last year, we isolated and purified a candidate ocular commensal, Corynebacterium mastitidis. That this organism elicited a commensal-specific interleukin 17 response from T cells in the ocular mucosa, tuning local host defense to afford protection from infectious pathogenic organisms (St. Leger et al., Immunity Aug 2017). We are now examining commensal-elicited responses in an immunologically abnormal host. In collaboration with the group of Dr. Warren Strober (NIAID) who developed knock-in mice expressing a gain-of-function mutation in the NLRP3 inflammasome gene, and Gabriela Goldbach Mansky who treats patients with NLRP3 inflammasome mutations, we have preliminary evidence to suggest that, in a host with an overactive inflammasome, an ocular surface commensal may elicit ocular surface inflammation, thus acting as a pathobiont.